ABSTRACT Salvia divinorum a member of the mint family, is a potent hallucinogen rapidly increasing in popularity as a drug of abuse. Though the abuse of illicit stimulants is a growing national problem, addiction treatments are unavailable. Salvinorin A (SalA), the primary psychoactive constituent of Salvia divinorum, is a highly potent kappa-opioid receptor (KOR) agonist. KOR systems have been implicated in depression pathogenesis and the mood dysregulation charactizing drug withdrawal. Despite on-going efforts to develop orally effective SalA analogues and other KOR ligands for depression and addiction treatments, the downstream effectors upon which SalA/KOR act to affect behavior are unknown. SalA and its analogues have therapeutic potential as mood and substance abuse treatments but are limited by their adverse aversive and dysphoric effects. The dysphoric and aversive effects of SalA have been attributed to decreased dopamine (DA) and serotonin (5-HT) release in the neucleus accumbens (NAc). 5-HT plays an important role in regulating the KOR systems impotantly, KOR proteins are expressed on serotonergic neurons and terminals. Presynaptic 5-HT transporter (SERT) controls serotonergic neurotransmission by rapid reuptake of released 5-HT, thus playing an important role in several behavioral and physiological functions. Our studies showed that depending on the duration of SalA exposure, SalA/KOR activaton differentially affects SERT function associated with sequential SERT phosphorylation. Acute exposure of SalA decreased SERT function via Thr276 phosphorylation while prolonged and/or repeated SalA exposure increased SERT function via Ser277 phosphorylation. Importantly, TAT-SERT peptide encompassing Thr276-Ser277 prevented the SalA-triggered biphasic mode of SERT regulation. These findings identify a novel mechanism by which SalA regulates presynaptic 5-HT transmission, suggesting that SERT dysregulation/phosphorylation may be one mechanism underlying SalA-mediated alterations in mood and affect. The studies in this proposal will test the hypothesis that: SalA/KOR activation modulates behaviors via biphasic regulation of SERT functions associated with sequential phosphorylation of Thr276?Ser277 motifs. Aim 1 will identify the cellular mechanisms of SalA/KOR-linked SERT modulation in vivo by determining whether SalA-mediated biphasic changes in SERT function in the NAc are associated with sequential Thr276?Ser277 phosphorylation. Aim 2 will determine the physiological relevance of SalA- triggered biphasic SERT modulation/phosphorylation to the behavioral effects of SalA. Findings from these studies will enhance our understanding of the neural substrates upon which SalA act to regulate behaviors, aid in the development of effective pharmacological agents for the treatment of addiction, depression and provide new insights into the role of SERT phosphorylation in regulating synaptic 5-HT clearance and behavior.